Our overall goal is to elucidate the molecular details of protein translocation across biological membranes using Escherichia coli as a facile genetic and biochemical system. We will focus on a central component, SecA ATPase, which interacts with all key components, and whose translocation ATPase activity and membrane cycling behavior underlie the energetics and translocation mechanism. Three specific aims are proposed. (1) To understand the molecular basis for SecA-signal peptide recognition, the SecA signal peptide-binding site will be mapped by fluorescence resonance energy transfer methodology, and competition and mutational studies will be undertaken to confirm the binding site assignment and elucidate the molecular modes responsible for degenerate but specific peptide binding in this system. (2) To understand the structural and functional bases of SecA-SecYEG interaction and SecA membrane cycling, secA mutants defective in these processes will be isolated and characterized, and in vivo SecA membrane topology studies will be employed to pinpoint SecYEG channel-accessible regions around the SecA ATP- binding site and amino-terminal extension arm that control SecA membrane cycling. (3) To elucidate the SecA ATPase reaction cycle, pre-steady-state and steady-state analyses of a SecA-SecYEG proteoliposome complex under "idling" and actively-translocating conditions will be undertaken. Parallel studies will also be undertaken with functional, mutant SecA proteins with remarkably reduced translocation ATPase activity. These studies should provide novel insights into the stepwise operation of the SecA nanomotor and its chemo-mechanical coupling to preprotein recognition, SecYEG channel activation and processive protein translocation. They should lead to a detailed understanding of Sec-dependent protein translocation at the molecular level, and they should be of broad significance to understand these processes in parallel systems, to engineer such pathways, and to develop novel anti-bacterial agents.